Insecticidal properties of Bacillus thuringiensis var. Israelensis. II. Comparative morphological and molecular genetic analysis of the crystallogenic and acrystallogenic strains

Currently, the bacterium Bacillus thuringiensis var. israelensis represents a key agent for biological protection against dipteran species, which are harmful to livestock and crop production and transmit infectious diseases of economically important animals. The production strains can be obtained by isolation from natural resources, selection of previously used isolates, screening of genetic collections, and genetic or genomic engineering. The issue of preservation and control of practically valuable properties of strains is of high importance. Biologicals are of significant interest due to their substantial advantages over chemical pesticides and are considered in modern agricultural systems as environmentally and socially priority alternatives to agrochemicals. In the present work, we performed the first comprehensive comparative analysis of crystallogenic and acrystallogenic variants of Bacillus thuringiensis var. israelensis (BtH14) isolated after storage of the strain in different modes. For crystallogenic variants, genes encoding the target insecticidal toxins, Cry4 and Cry11, were detected by the polymerase chain reaction (PCR), and it was shown that the acrystallogenic variants are devoid of these genes. It was found that the culture fluid of crystallogenic variants is approximately 7000 times more active against the Aedes aegypti larvae than the same of acrystallogenic. The aim of this work was to compare the morphological, biochemical, technological, larvicidal properties of the crystal-forming and acrystallogenic variants of the strains of Ваcillus thuringiensis var. israelensis (BtH14) and testing for the presence of genes encoding Cry insecticidal toxins, which are key determinants of virulence. We studied the strains 404 and 87 stored for 28 years by freeze-drying, then 2 years in test tubes on canted fish agar (FA) with replanting every 6 months; the 7-1/23 strain stored for 28 years in crystals of NaCl, then 2 years in culture liquid (CL) at 3 °С. Bacterial strains were inoculated on Petri dishes to obtain separate colonies. On day 7 of growth, the 404/14, 87/21, 7-1/23-4 (crystal-forming) and 404/19, 87/33, 7-1/23-8 (acrystallogenic) variants were selected by microscopic analysis using aniline black dye. The differences in the colony morphology were not revealed: the colonies were flat, opaque, grayish-white, rough, rounded, the structure was fine-grained, and the consistency was viscous. The differences either in the morphology of the vegetative cultures, or in the main biochemical properties (the formation of acetylmethyl carbinol, lecithinase, the use of carbohydrates, the splitting of starch, etc.), or in the titer on the yeast-polysaccharide medium were not shown as well. The productivity of the 404/14, 87/21, 7-1/23-4 and 404/19, 87/33, 7-1/23-8 strains varied from 3.36×109 CFU/ml to 4.02×109 CFU/ml and from 3.74×109 CFU/ml to 4.13×109 CFU/ml, respectively. The larvicidal activity of the crystal-forming variants, expressed in LC50 for L4 Aedes aegypti , was (0.12-0.16)×10-3 %, while acrystallogenic variants were inactive within the standard dilutions (×10-3 %) 1.0; 0.5; 0.25; 0.125; 0.06. Only their 1 % suspension (7000-fold higher concentration) caused 22-39 % death of the Aedes larvae after 24 hours; the same concentration of active variants resulted in 100 % death in 15 minutes. It was established that cultural liquid of the acrystallogenic variants formed a precipitate and a supernatant layer after 12 hours, while the crystal-forming variants remained suspended. The investigated variants of BtH14 were analyzed for the presence of genes encoding insecticidal toxins. The results of the PCR analysis with the Bti-specific primers confirmed the belonging of the both crystal-forming and acrystallogenic variants to BtH14. It has been found that the 404/14, 87/21, 7-1/23-4 strai